突变k-ras基因重组腺病毒的构建

目的　利用细菌内同源重组法构建含 1 2密码子点突变的k ras基因重组腺病毒。方法　用限制性内切酶kpnⅠ +xhoⅠ从载体pcDNA3 k ras1 2 (Val)中切出 1 2密码子点突变的k ras基因片断 ,亚克隆至经同样酶切的腺病毒穿梭质粒pAdTrack CMV中 ,形成转移质粒pAdTrack CMV/k ras 1 2 (Val) ,将之PmeⅠ酶切线性化后与腺病毒基因重组质粒pAdEasy 1共转化大肠杆菌BJ51 83 ,抽提经鉴定含目的基因的重组体质粒 ,PacⅠ酶切后用脂质体转染 2 93细胞 ,包装成重组体腺病毒Ad k ras1 2 (Val)。采用PCR方法对重组体腺病毒进行鉴定 ,利用穿梭质粒pAdTrack CMV中带有GFP报告基因 ,对病毒滴度和感染效率进行监测。结果　利用CaCl2 法由pAdTrack CMV/k ras 1 2 (Val)和pAdEasy 1共转化大肠杆菌BJ51 83 ,可获得 2 5%左右的阳性重组体细菌克隆。PCR检测表明重组腺病毒已含有目的基因 ,滴度为 1 .2× 1 0 1 2 pfu/ml。结论　细菌内同源重组法构建腺病毒相比于传统的细胞内同源重组法 ,具有成功率高、方法简便、快捷、实验周期短的优点 ,值得进一步推广使用     

简化的两步法细菌内同源重组高效制备腺病毒质粒

目的：应用简化的两步法细菌内同源重组高效制备腺病毒质粒。方法：对细菌内同源重组法进行改进和简化，先构建含腺病毒基因组质粒pAdEasy-1的BJ5183细菌，筛选出链霉素和氨苄青霉素抗性菌落，继而应用氯化钙法制作BJ5183pAdEasy-1感受态细菌。用PmeⅠ酶使转移质粒pAdtrack-CMV-TK线性化，和BJ5183pAdEasy-1感受态细菌混合进行转化，在含12．5μg／mL卡那霉素的LB琼脂平皿上培养，筛选出卡那霉素抗性的细菌进行质粒抽提纯化，获得重组腺病毒质粒。结果：卡那霉素抗性细菌有2种，一种是含pAdeasy-CMV-TK(约34kb)，另一种含pAdtrack-CMV-TK(约10kb)，两者可经琼脂糖电泳加以鉴别。构建重组腺病毒质粒的成功率达90％(9／10)。结论：简化的两步法细菌内同源重组是一种简便易行、快速高效的腺病毒质粒构建方法。     

SLC-Her-2/neu-P53-Fc融合基因重组腺病毒表达载体的构建及检测

目的构建携带融合基因SLC-Her-2/neu-P53-Fc(SLC-HP-Fc)的重组腺病毒AdEasyTM-SLC-HP-Fc表达载体。方法提取SLC-HP基因及含Fc段基因的质粒构建携带目的基因的腺病毒穿梭载体pShuttle-CMVSLC-HP-Fc,经酶切线性化后转入含有pAdEasy-1质粒的E.coli BJ5183中进行同源重组,得到重组腺病毒真核表达载体AdEasyTM-SLC-HP-Fc。线性化后的病毒表达载体通过脂质体Lipofectamine 2000转染293细胞,通过包装扩增并经PCR鉴定后得到大量复制病毒并纯化保存。结果得到重组腺病毒AdEasyTM-SLC-HP-Fc,成功转染293细胞出现细胞病变反应,经酶切及PCR法确定目的基因的表达。结论成功构建了SLC-Her-2/neu-P53-Fc融合基因重组腺病毒表达载体,并建立了重组腺病毒AdEasyTM-SLC-HP-Fc种子病毒库和工作病毒库,为进一步研究该融合基因与肿瘤相关作用奠定了实验基础。     

简化的两步法细菌内同源重组高效制备腺病毒质粒

目的 :应用简化的两步法细菌内同源重组高效制备腺病毒质粒。方法 :对细菌内同源重组法进行改进和简化 ,先构建含腺病毒基因组质粒pAdEasy 1的BJ5 183细菌 ,筛选出链霉素和氨苄青霉素抗性菌落 ,继而应用氯化钙法制作BJ5 183pAdEasy 1感受态细菌。用PmeⅠ酶使转移质粒pAdtrack CMV TK线性化 ,和BJ5 183pAdEasy 1感受态细菌混合进行转化 ,在含 12 5 μg mL卡那霉素的LB琼脂平皿上培养 ,筛选出卡那霉素抗性的细菌进行质粒抽提纯化 ,获得重组腺病毒质粒。结果 :卡那霉素抗性细菌有 2种 ,一种是含pAdeasy CMV TK(约 34kb) ,另一种含pAdtrack CMV TK(约 10kb) ,两者可经琼脂糖电泳加以鉴别。构建重组腺病毒质粒的成功率达 90 % (9 10 )。结论 :简化的两步法细菌内同源重组是一种简便易行、快速高效的腺病毒质粒构建方法     

HBx基因重组腺病毒载体构建及其在SMMC-7721细胞中的表达

目的:利用pAdEasy1腺病毒载体系统构建人HBx基因重组腺病毒,感染肝癌细胞株SMMC7721使HBx基因有效表达。方法:自真核表达载体pcDNA3.1(-)HBx中获得HBx基因,插入腺病毒穿梭质粒pAdTrackCMV中构建pAdTrackCMVHBx,然后经PmeⅠ酶切线性化,电转化到含腺病毒骨架质粒pAdEasy1的BJ5183感受态细菌中。挑选和鉴定正确的同源重组质粒,然后将线性化的重组质粒转染293N细胞,产生重组病毒颗粒,并进一步感染SMMC7721细胞株。结果:经限制性内切酶酶切和基因测序鉴定,证实pAdTrackCMVpAdEasy1HBx重组成功,借助荧光显微镜可以观察到绿色荧光蛋白GFP在293N和SMMC7721细胞株中表达。结论:成功构建了携带HBx基因的重组腺病毒载体,并在SMMC7721细胞中使HBx基因有效表达,为后续研究奠定了基础。     

携带凋亡素基因重组腺病毒的制备及诱导结肠癌细胞凋亡的实验研究

摘 要：［目的］ 利用细菌内同源重组法构建携带凋亡素VP3基因的重组腺病毒并观察其体外诱导结肠癌细胞凋亡的作用。［方法］ 设计VP3 cDNA扩增引物,从PET15b-VP3质粒中扩增VP3的DNA序列,与线性pShuttle-IRES-hrGFP连接构建pShuttle-VP3-hrGFP重组穿梭质粒,PCR和EcoR Ⅴ酶切电泳鉴定;重组穿梭质粒经Pmel酶切线性化后,转化含pAdeasy-1的超感受态BJ5183大肠杆菌,细菌内同源重组法构建重组腺病毒质粒pAd-VP3-hrGFP,PCR、PacI酶切电泳及测序鉴定;线性化重组腺病毒质粒经脂质体转染AD293细胞进行pAd-VP3-hrGFP重组腺病毒的包装和扩增,CsCI密度梯度离心法进行病毒浓缩和纯化并将其作用人结肠癌SW480细胞,观察其对细胞凋亡及周期分布的影响。［结果］ pShuttle-VP3-hrGFP重组穿梭质粒构建成功;pAd-VP3-hrGFP重组腺病毒质粒经酶切获得一大于23kb的大片段和4.5kb的片段,PCR反应扩增出402bp的片段,测序证实VP3-hrGFP编码区成功克隆入腺病毒pAd中,且其序列与GeneBank中VP3序列完全一致;成功包装出携带VP3基因的腺病毒,滴度为1.738×1012opu/ml,重组腺病毒可阻滞结肠癌细胞周期于G0/G1期,诱导细胞凋亡,MOI值越大作用效应越显著(P<0.05,P<0.01)。［结论］ 细菌内同源重组法可快速、高效制备携带凋亡素基因的重组腺病毒,并具有较强的抗肿瘤作用,为深入研究VP3基因功能提供了选择。     

Smad3D和Smad7重组腺病毒载体的快速构建

目的 简化重组腺病毒载体的构造方法，构建含Smad3DcDNA或Smad7cDNA的重组腺病毒载体，为下一步的基因治疗奠定基础。方法 以AdEasy System为基础，应用序贯化学转化方法，在大肠杆菌E．coli BJ5183体内将携带目的基因的穿梭质粒和骨架质粒重组。结果 成功构建了重组腺病毒载体pAd-Smad3D和pAd-Smad7及空腺病毒载体，并经酶切鉴定证实。结论 用序贯化学转化方法，可以快速高效地在大肠杆菌内构建重组腺病毒载体。     

细菌内同源重组法高效制备含CD、TK融合自杀基因重组体腺病毒

目的：采用细菌内同源重组法高效制备含ＣＤ、ＴＫ融合自然基因重组体腺病毒。方法：ＣＤ、ＴＫ融合基因ＣＤｇｌｙＴＫ自载体ｐＷＺＬｎｅｏＣＤｇｌｙＴＫ中切出,亚克隆至腺病毒穿梭质粒中,形成转移质粒ｐＡｄｔｒａｃｋＣＭＶ－ＣＤｇｌｙＴＫ,将之Ｐｍｅｌ酶切线性化后与腺病毒基因组质粒ｐＡｄｅａｓｙ０１共转化ＢＪ５１８３菌,抽提重组体腺病毒基因组质粒ＤＮＡ,Ｐａｃｌ酶切后转染２９３细胞包装成腺病毒颗粒,采用ＰＣ     

沉默结缔组织生长因子基因重组腺病毒的构建及功能验证

目的:构建沉默结缔组织生长因子(connective tissue growth factor, CTGF)基因的复制缺陷型重组腺病毒Ad-siCTGF,并进行功能验证.方法:选取已验证的能高效沉默CTGF基因的靶序列,克隆入载体pSES-HUS中;将质粒线性化处理后与腺病毒骨架质粒pAdEasy共同转染大肠埃希菌BJ5183,构建重组腺病毒载体Ad-siCTGF;腺病毒载体用Pac I酶切线性化处理后转染HEK293细胞,包装重组腺病毒;采用"乒乓交互感染法"提高病毒滴度.用此病毒感染4T1细胞,行实时荧光定量PCR(real-time fluorescence quantitative -PCR, RFQ-PCR)及Western 印迹法验证其沉默效果.结果:Pac I酶切电泳证实重组腺病毒载体Ad-siCTGF构建成功,扩增纯化后测定重组病毒Ad-siCTGF的滴度为2.6×10~(10) pfu/mL.感染此病毒后,4T1细胞中CTGF mRNA表达水平下调至36.27%,蛋白表达水平下调至31.56%.结论:成功构建能沉默CTGF基因的重组腺病毒,为进一步研究CTGF在肿瘤中的作用机制奠定了基础.     

介导尤文肉瘤融合基因的重组腺病毒的构建及其在外周血单个核细胞(PBMC)中的表达

目的　构建尤文肉瘤融合基因EWS -FLI1重组腺病毒 ,并检测其在外周血单个核细胞(PBMC)中的表达 ,为进一步进行尤文肉瘤的免疫治疗奠定基础。方法　将质粒pEC1EWS/FLI1酶切 ,切出的EWS -FLI1cDNA片段克隆至腺病毒穿梭质粒padtrack -cmv的HCMV启动子下游。将连接后的穿梭质粒和骨架质粒Padeasy -1共同转化大肠杆菌BJ5 1 83菌株 ,获得同源重组后的腺病毒质粒pADEWS/FLI1。将此质粒转染 2 93细胞 ,包装产生腺病毒AdEWS -FLI1。扩增、纯化产生高滴度的AdEWS -FLI1。转染PBMC ,并通过RT -PCR和免疫组化法检测EWS -FLI1的表达。结果　同源重组后产生的pADEWS/FLI1经PCR鉴定构建成功 ,纯化后的滴度为 4× 1 0 10 /ml。转染PBMC后 ,RT -PCR证实有EWS/FLI1mRNA的转录 ,免疫检测法检测在PBMC中有EWS/FLI1的表达。结论　重组腺病毒AdEWS/FLI1构建成功 ,并能在PBMC中稳定有效地表达 ,为进一步进行尤文肉瘤的免疫治疗研究奠定了基础。     

Leukaemogenic and mutagenic activity of the butyrophenon, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (haloperidol)

Development of mainly monocytic-myeloid leukaemias was observedin two strains of mice upon i.p. administration of 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone(haloperidol), an antipsychotic drug. Haloperidol was also shownto be mutagenic in the Ames Salmonella test system.     

Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formed metabolically from 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK). Using the chiral derivatizing agent, (R)- (+)-alpha-methylbenzyl isocyanate [(R)-(+)-MBIC], previous work has shown that the enantiomeric ratio of metabolically formed NNAL and its glucuronide derivative may be species dependent. However, the absolute configuration of such NNAL has not been previously reported. Synthetically prepared racemic NNAL was converted to diastereomeric esters by reaction with (R)-(+)- and (S)-(-)-alpha-methoxy-alpha- (trifluoromethyl)phenylacetic acid (MTPA) chloride (Mosher's reagent) and the products were characterized by 1H-NMR. Based on chemical shift data, the absolute configuration of NNAL in each diastereomeric ester was assigned. Hydrolysis of (R)-NNAL-(R)-MTPA gave (R)-NNAL. This was converted to the corresponding carbamate by reaction with (R)-(+)-alpha- MBIC and the absolute configurations of the diastereomeric carbamates formed by reaction of (R)- and (S)-NNAL with (R)-(+)-MBIC were thereby assigned. Conversion of metabolically produced NNAL to the same carbamates allowed us to assign the NNAL formed from NNK by rat liver microsomes as (R)-NNAL. The major and minor NNAL-glucuronide diastereomers found in the urine of patas monkeys and humans exposed to NNK were similarly assigned; they were formed from (R)-NNAL and (S)- NNAL, respectively.      

Polyamine analog bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) enhances simian virus 40 late gene expression

Purpose: The polyamine analog bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) depletes cellular polyamines and inhibits malignant cell growth. We have previously shown that BE-4-4-4-4 inhibits nucleosome condensation on supercoiled DNA in a cell-free system. Here we sought to determine whether BE-4-4-4-4 inhibits nucleosome condensation in cells, and whether that effect alters the expression of specific genes. Methods: We used the simian virus 40 (SV-40) minichromosome as a model system and studied the expression of the viral late genes. It is known that the SV-40 late genes are regulated by the steroid receptor elements that, in turn, control gene expression by altering nucleosomal organization. Results: We observed a more than six fold increase in SV-40 late gene expression in cells pretreated with BE-4-4-4-4 for 18 h. The polyamine analog bisethyl norspermine (BE-3-3-3), that does not affect nucleosomal condensation in cell free systems and has little effect on chromatin structure in cultured human tumor cells, had a negligible effect on SV-40 late gene expression under treatment conditions identical to those used with BE-4-4-4-4. Conclusion: Similar to the findings in the cell-free system, the polyamine analog BE-4-4-4-4 inhibited nucleosome formation and, thereby, altered the expression of specific genes in a cellular system. Received: 4 May 1998 / Accepted: 24 July 1998     

Tumorigenic activity of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine (NNN) on topical application to Sencar mice

The tumor-initiating activities of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine (NNN) were evaluated on the skin of female SENCAR mice. A total initiator dose of 28 mumol/mouse of each nitrosamine was applied in 10 subdoses administered every second day. Promotion commenced 10 days after the last initiator dose and consisted of twice weekly application of 2.0 micrograms of tetradecanoylphorbol acetate for 20 weeks. NNK induced a 79% incidence of skin tumors with an average of 1.6 tumors/mouse and a 59% incidence of lung adenomas. In contrast, iso-NNAL and NNN were not active as tumor initiators in either the skin or lung of mice. The tumorigenic activity of NNK on SENCAR mouse skin was evaluated at several doses. At a total initiator dose of 28 and 5.6 mumol/mouse, NNK exhibited significant activity (P less than 0.005) inducing a 59% and 24% incidence of skin tumors, respectively. In this dose response bioassay, NNK at a total initiator dose of 28 mumol induced a 63% incidence (P less than 0.005) of lung adenomas. The numbers of lung adenomas induced at the lower doses employed were not significant. NNK, at a total initiation dose of 1.4 mumol, did not exhibit significant tumorigenic activity (P greater than 0.05). Analysis of DNA from the skin of mice treated with NNK using HPLC with fluorescence detection failed to detect O6- and N-methylguanine (O6-MG and N7-MG) adducts. These data indicate that NNK can exert a contact carcinogenic effect and suggest that mechanisms other than DNA methylation may be involved in its activation to a tumorigenic agent in mouse skin.     

Comparative mutagenicity of 4-(carbethoxynitrosamino)-4-(3-pyridyl)butanal and 4-(carbethoxynitrosamino)-1-(3-pyridy1)-1-butanone, model compounds for {alpha}-hydroxylation of N'-nitrosonornicotine

4-(Carbethoxynitrosamino)-1-(3-pyridy1)butanal, a stable precursorto the putative diazohydroxide formed by 5'-hyd-roxylation ofthe tobacco-specific nitrosamine, N'-nitrosonornicotine, wassynthesized in six steps from nicotinaldehyde. Its mutagenicitytoward S. typhimurium was compared to that of 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone,a precursor to the diazohydroxide formed by 2'-hydroxylationof N'-nitrosonornicotine. At equimolar doses, 4-(carbethoxy-nitrosamino)-1-(3-pyridy)-1-butanonewas a potent mutageon, but 4-(carbethoxynitrosamino)-1-(3-pyridy)butanal was inactive toward strains TA 100 and TA 1535. Theresults of this study indicate that the putative diazohydroxideformed by 2'-hydroxylation of N'-nitrosonornocotine has higherinherent mutagemicity toward S. typhimurinum than does the correspondingdiazohydroxide formed by 5'-hydroxylation.     

Genetic variability in the metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)

Urinary metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides, termed total NNAL, have recently been shown to be good predictors of lung cancer risk, years before diagnosis. We sought to determine the contribution of several genetic polymorphisms to total NNAL output and inter-individual variability. The study subjects were derived from the Harvard/Massachusetts General Hospital Lung cancer case-control study. We analyzed 87 self-described smokers (35 lung cancer cases and 52 controls), with urine samples collected at time of diagnosis (1992-1996). We tested 82 tagging SNPs in 16 genes related to the metabolism of NNK to total NNAL. Using weighted case status least squares regression, we tested for the association of each SNP with square-root (sqrt) transformed total NNAL (pmol per mg creatinine), controlling for age, sex, sqrt packyears and sqrt nicotine (ng per mg creatinine). After a sqrt transformation, nicotine significantly predicted a 0.018 (0.014, 0.023) pmol/mg creatinine unit increase in total NNAL for every ng/mg creatinine increase in nicotine at p < 10E-16. Three HSD11B1 SNPs and AKR1C4 rs7083869 were significantly associated with decreasing total NNAL levels: HSD11B1 rs2235543 (p = 4.84E-08) and rs3753519 (p = 0.0017) passed multiple testing adjustment at FDR q = 1.13E-05 and 0.07 respectively, AKR1C4 rs7083869 (p = 0.019) did not, FDR q = 0.51. HSD11B1 and AKR1C4 enzymes are carbonyl reductases directly involved in the single step reduction of NNK to NNAL. The HSD11B1 SNPs may be correlated with the functional variant rs13306401 and the AKR1C4 SNP is correlated with the enzyme activity reducing variant rs17134592, L311V.     

Identification and analysis of a new tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol

A new tobacco-specific N-nitrosamine, 4-(methylnitros-amino)-4-(3-pyridyl)-1-butanol(iso-NNAL) was isolated from snuff tobacco. Structural characterizationof this N-nitros-amine was confirmed by mass spectral analysis.Five popular US brands of moist snuff and three popular US brandsof dry snuff tobacco were analyzed for moisture, nicotine andtobacco-specific N-nitrosamines. The moisture content variedfrom 20 to 53% in moist snuff and from 4.7 to 5.6% in dry snuff.The nicotine levels in these samples varied from 0.6 to 3.2%.The newly identified iso-NNAL was present in con centrationsranging from 0.07 to 2.5 p.p.m. whereas other tobacco-specificN-nitrosamines, N-nitrosonornicotine, N-nitrosoanatabine, N-nitrosoanabasineand 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanone were foundto range from 0.1 to 178 p.p.m. Iso-NNAL was not detected inmainstream and sidestream smoke of cigarettes. Iso-NNAL is genotoxicin primary rat hepatocytes; its tumorigenic properties are cur-rentlybeing tested in mice and rats.     

DNA and hemoglobin alkylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanol in F344 rats

Alkylation of DNA and hemoglobin was compared in male F344 ratsgiven a single s.c. injection of the tobacco-specific nitrosamine4-(methyInitrosamino)-1-(3-pyridyl)-1-butanone (NNK), or itsmajor metabolite formed by carbonyl reduction, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL).In hepatic DNA, levels of 7-methylguanine and O⁶-methyl-guanineformed from NNK 1-48 h after treatment were similar to thoseformed from NNAL. In nasal mucosa and lung DNA, levels of 7-methylguanineand O⁶Amethylguanine were somewhat higher after treatment withNNK than with NNAL. Acid hydrolysis of hepatric DNA, isolatedfrom rats treated with either [5-³H]NNK or [5-³H]NNAL, gave180 ± 48 or 120 ± 23 µuno/mol guanine, respectively,of 4-hydroxy-1-(3-pyridyl)-1-butanone. Basic hydrolysis of globinisolated from rats treated with either [5-³H]NNK of 5-³H]NNALgave 4.1 ± 0.7 or 2.0 ± 0.1 pmol/mg, respectivelyof 4-hydroxy-1-(3-pyridyl)-1-butanone. These results indicatethat NNAL is not a detoxification product of NNK, since treatmentof rats with NNAL results in modifications of DNA which arequalitativerly and quantitatively similar to those observedupon treatment with NNK. Alkylation of DNA and globin by NNALmay result mainly from its metabolic reconversion to NNK.